Molecular and Nano-Structural Optimization of Nanoparticulate Mn2+-Hexarhenium Cluster Complexes for Optimal Balance of High T1- and T2-Weighted Contrast Ability with Low Hemoagglutination and Cytotoxicity

The present work introduces rational design of nanoparticulate Mn(II)-based contrast agents through both variation of the μ3 (inner) ligands within a series of hexarhenium cluster complexes [{Re6(μ3-Q)8}(CN)6]4− (Re6Q8, Q = S2−, Se2− or Te2−) and interfacial decoration of the nanoparticles (NPs) K4−2xMnxRe6Q8 (x = 1.3 − 1.8) by a series of pluronics (F-68, P-123, F-127). The results highlight an impact of the ligand and pluronic for the optimal colloid behavior of the NPs allowing high colloid stability in ambient conditions and efficient phase separation under the centrifugation. It has been revealed that the K4−2xMnxRe6Se8 NPs and those decorated by F-127 are optimal from the viewpoint of magnetic relaxivities r1 and r2 (8.9 and 10.9 mM−1s−1, respectively, at 0.47 T) and low hemoagglutination activity. The insignificant leaching of Mn2+ ions from the NPs correlates with their insignificant effect on the cell viability of both M-HeLa and Chang Liver cell lines. The T1- and T2-weighted contrast ability of F-127–K4−2xMnxRe6Q8 NPs was demonstrated through the measurements of phantoms at whole body 1.5 T scanner.


Introduction
Paramagnetic enhancement of transverse and longitudinal magnetic relaxation rates of water protons in aqueous solutions of ions, or complexes of paramagnetic transition metals, provides contrast-enhancement of MRI. Gd(III)-based contrast agents (CAs) are the best from the viewpoint of the accelerating of the relaxation processes, however, their side effects [1] prompt rapidly growing interest to more biogenic Mn(II)-based CAs.

Methods
The detailed description of the common methods (dynamic light scattering (DLS), powder X-ray diffraction (PXRD), inductively coupled plasma optical emission spectrometry (ICP-OES), transmission electron microscopy (TEM), UV-Vis, IR and electronic spin resonance spectroscopy) are in the Supplementary Materials.

Relaxometry
The proton relaxation times T 1 and T 2 were measured using pulsed NMR-relaxometer Minispec MQ20 from Bruker with operational frequency of 19.65 MHz (0.47 T) by applying the standard radio frequency pulse sequences: inversion-recovery method (spin-lattice relaxation time T 1 ), and Carr-Purcell sequence, modified by Meiboom-Gill (spin-spin relaxation time T 2 ) with the measuring accuracy error smaller than 3%. The temperature was maintained with the Thermo/Haake DC10 circulator.
The contrasting ability of the as-prepared Mn-containing colloids was demonstrated through the T 1 -and T 2 -weighted images obtained by means of whole body 1.5 T scanner (Excel Art Vantage Atlas X, Toshiba, Otawara, Japan) equipped with 65-cm horizontal bore size corresponding to a proton resonance frequency of 63.58 MHz. The detailed description of the measuring procedure is in the Supplemetary Materials. The as-synthesized Mn x Re 6 Q 8 NPs were precipitated in a centrifuge at 14,500 rpm for 40 min at a temperature of 310-318 K. The separated NPs were redispersed in 3.4 mL of the pluronic's solutions (1 g·L -1 ) through the ultrasonication for 10 min at 288 K.

Determination of Re:Mn Ratio
The spectrophotometric determination of Mn 2+ content in the supernatants is based on its complexation with xylenol orange (XO) [39,40] at pH = 6.8, which leads to increase of intensity of the absorption band at 580 nm. The standard solution of XO (1 mM) was prepared (see ESI). Three solutions were prepared: 1-reference solution of XO (2.9 mL of phosphate buffer + 0.1 mL of XO); 2-measurement solution (2.8 mL of phosphate buffer + 0.1 mL of XO + 0.1 mL of the supernatant, obtained after precipitation of Mn x Re 6 Q 8 ); 3-reference solution of the Mn-XO complex (2.8 mL of phosphate buffer + 0.1 mL of XO + 0.1 mL of MnCl 2 (0.588 mM), which simulate 100% manganese in the system). UV-vis spectra were recorded for all solution ( Figure S1).
The residual amount of Mn 2+ (%) in the supernatant was determined by the formula: The concentration of manganese in the particles was determined as C max * (100-C(st)), where C max is concentration of Mn 2+ in initial solution before centrifugation.  Figure S2).

Hemagglutination Assay
Human erythrocytes were washed twice with physiological saline (NaCl 0.9%) and centrifuged at 2500× g for 10 min at 4 • C. After each cycle, the supernatant was carefully removed. Then the red blood cells were resuspended in physiological saline to a concentration of 2%. Hemagglutination activity was analyzed in a 96-well U-plate. Double dilution series of the aqueous colloids of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 were prepared. 100 µL of the colloids was mixed with 100 µL of 2% red blood cell solution and put into a well. Each concentration point was carried out simultaneously in two wells in parallel. After 1 h of incubation at 37 • C, hemagglutination was observed with unaided eye [41] and checked using Nikon Eclipse Ci-S fluorescence microscope (Nikon, Nasu Nasu-Gun, Japan) with Ph1 condenser ring in phase contrast mode. A suspension of red blood cells in 0.9% saline and a mixture of type A(II) and C(IV) erythrocytes were used as negative and positive agglutination control, respectively.

Synthesis and Characterization of K 4−2x Mn x Re 6 Q 8
Literature data indicate that coordination of Mn 2+ ions with [{Re 6 Q 8 }(CN) 6 ] 4− (Re 6 Q 8 , Q = S 2− , Se 2− or Te 2− ) generates great diversity of MOF-like structures, where the framework derives from Re-C-N-Mn bridges, while the binding still remains the opportunity of the Mn 2+ ions to coordination of water, solvent molecules or chelating ligands [27]. According to these reports the mixing of manganese salts with potassium salts of [{Re 6 Q 8 }(CN) 6 ] 4− in aqueous solutions triggers a formation of MOF-like supramolecular structures, which can be applied in generation of MOF-based NPs under the conditions of controlling the size of the formed NPs. The facile route to control the size was previously demonstrated for the MOF-based nanoparticles constructed from the [{Re 6 Q 8 }(CN) 6 ] 4− and Gd 3+ ions with the use of F-127 as hydrophilic agent [42]. However, problems with a phase separation of ultra-small NPs coated by hydrophilic shell indicate a necessity for a balance between high colloid stability of hydrophilic NPs and their ability to easy phase separation. The use of triblock copolymers as hydrophilic agents provides an ability to control colloidal behavior of the Mn(II)-based NPs by managing of their hydrophilic shell.
The structure diversity of triblock copolymers derived from the lengths' variation of PEO and PPO blocks allows to modify their self-aggregation in aqueous solutions. The Mn(II)-based NPs were obtained for [{Re 6 Figure 1a along with the most probable structural motifs of the binding of the Mn 2+ ions with the cluster units (Figure 1b,c). The comparative analysis of the synthetic data at various structure and concentration of the components reveals the conditions optimal for formation and separation of the NPs, which were further characterized by elemental analysis, TEM, PXRD, DLS techniques ( Table 1).
The stoichiometry of the heterometallic NPs was determined through both spectrophotometric XO-assisted analysis of supernatants and ICP-OES analysis of the separated colloids (Table S1). The Mn:Re 6 ratios determined by the methods are very close, indicating that the values are about 1.3, 1.8 and 1.8 for Re 6 Q 8 at Q = S 2− , Se 2− and Te 2− , respectively (Table 1). This confirms heterometallic nature of the NPs, which herein and further will be designated as K 4−2x Mn x Re 6 Q 8 . the aggregation is significantly greater for P-123 ( Table 1). The concentration of the triblock copolymers at the level of 1 g•L −1 corresponds to 0.08 mM of F-127, 0.12 mM of F-68 and 0.17 mM of P-123. Thus, only P-123 forms the micellar aggregates in the solutions at 295 K [37], while the CMC (critical concentration of micellization) values of F-127 and F-68 in these conditions are above the applied concentrations [43,44]. Thus, the triblock copolymer molecules should be "free" for interfacial stabilizing of the NPs, while the selfaggregation of the triblock copolymers decreases their capacity to hydrophilize the NPs. The aforesaid allows to choose F-127 and F-68 as more convenient hydrophilic agents than P-123, although the centrifugation-induced phase separation is very poor at ambient temperatures. It is worth noting the temperature-dependent micelle formation is the key specificity of the aggregation behavior of the triblock copolymers [37,44]. In particular, the  It is worth noting that F-127 and F-68 provide enough stabilization of the NPs, while the aggregation is significantly greater for P-123 ( Table 1). The concentration of the triblock copolymers at the level of 1 g·L −1 corresponds to 0.08 mM of F-127, 0.12 mM of F-68 and 0.17 mM of P-123. Thus, only P-123 forms the micellar aggregates in the solutions at 295 K [37], while the CMC (critical concentration of micellization) values of F-127 and F-68 in these conditions are above the applied concentrations [43,44]. Thus, the triblock copolymer molecules should be "free" for interfacial stabilizing of the NPs, while the selfaggregation of the triblock copolymers decreases their capacity to hydrophilize the NPs.
The aforesaid allows to choose F-127 and F-68 as more convenient hydrophilic agents than P-123, although the centrifugation-induced phase separation is very poor at ambient temperatures. It is worth noting the temperature-dependent micelle formation is the key specificity of the aggregation behavior of the triblock copolymers [37,44]. In particular, the CMC of F-127 decreases to 0.0028 mM at 303 K [45], while the temperature of micellization is above 323 K for F-68 (1 g·L −1 ) [44]. This indicates that the temperature rise under the centrifugation conditions can facilitate the phase separation of the NPs. Indeed, the temperature level at 303 K is enough to reach complete phase separation of the NPs from the F-127-based solutions under the centrifugation (for more details see Section 2), while the insufficient phase separation of the NPs is observed from the solutions of F-68 at the same temperature. Thus, the heating to 303 K decreases an extent of the "free" triblock copolymers due to their aggregation into micelles. This can restrict a participation of the triblock copolymers in hydrophilic coating of the NPs, thus, facilitating their phase separation under the centrifugation. In order to recognize a specific temperature-dependent effect of F-127 on the aggregation behavior of the NPs the latter was monitored at various temperatures in the solutions of P-123, F-68, F-127 (1 g·L −1 ). The DLS data ( Figure 2) reveal the detectable aggregation of the NPs in the solutions under their heating to 303 K for F-127 and P-123, which is enough for the centrifugation-induced phase separation of K 4−2x Mn x Re 6 Q 8 . The lack of detectable aggregation observed for F-68 correlates with the poor phase separation of the NPs in the same conditions. This confirms the aforesaid assumption that the ability of the NPs to phase separation can be controlled by the aggregation behavior of the triblock copolymers. It is worth noting that the developed synthetic procedure is reproducible (has been tested for six times at least), facile and not time consuming, in particular, the synthesis takes no more than 1.5 h. perature level at 303 K is enough to reach complete phase separation of the NPs from the F-127-based solutions under the centrifugation (for more details see Section 2), while the insufficient phase separation of the NPs is observed from the solutions of F-68 at the same temperature. Thus, the heating to 303 K decreases an extent of the "free" triblock copolymers due to their aggregation into micelles. This can restrict a participation of the triblock copolymers in hydrophilic coating of the NPs, thus, facilitating their phase separation under the centrifugation. In order to recognize a specific temperature-dependent effect of F-127 on the aggregation behavior of the NPs the latter was monitored at various temperatures in the solutions of P-123, F-68, F-127 (1 g•L −1 ). The DLS data ( Figure 2) reveal the detectable aggregation of the NPs in the solutions under their heating to 303 K for F-127 and P-123, which is enough for the centrifugation-induced phase separation of K4−2xMnxRe6Q8. The lack of detectable aggregation observed for F-68 correlates with the poor phase separation of the NPs in the same conditions. This confirms the aforesaid assumption that the ability of the NPs to phase separation can be controlled by the aggregation behavior of the triblock copolymers. It is worth noting that the developed synthetic procedure is reproducible (has been tested for six times at least), facile and not time consuming, in particular, the synthesis takes no more than 1.5 h. The separated colloids were dried and characterized by IR spectroscopy with the focus on the bands arisen from the apical cyanide ligands of the cluster complexes, since their binding with Mn 2+ ions should be followed by the shifting of maxima of the bands to higher energies as it was exemplified by [27]. Indeed, the shifting was revealed from the comparison the bands attributed to CN-groups in the K4−2xMnxRe6Q8 NPs with those of the corresponding K4[{Re6Q8}(CN)6] salts (Figures 3 and S3). This confirms that Mn 2+ ions are coordinated with the apical cyanides of the Re6Q8 cluster units. The residual amounts of F-127 molecules are also revealed from the IR spectra ( Figure S3). The separated colloids were dried and characterized by IR spectroscopy with the focus on the bands arisen from the apical cyanide ligands of the cluster complexes, since their binding with Mn 2+ ions should be followed by the shifting of maxima of the bands to higher energies as it was exemplified by [27]. Indeed, the shifting was revealed from the comparison the bands attributed to CN-groups in the K 4−2x Mn x Re 6 Q 8 NPs with those of the corresponding K 4 [{Re 6 Q 8 }(CN) 6 ] salts ( Figure 3 and Figure S3). This confirms that Mn 2+ ions are coordinated with the apical cyanides of the Re 6 Q 8 cluster units. The residual amounts of F-127 molecules are also revealed from the IR spectra ( Figure S3).  PXRD analysis of dried colloids reveals different extent of crystallinity of the colloids (Figure 4). The similarity in the PXRD patterns of K4−2xMnxRe6S8 and K4−2xMnxRe6Se8 colloids indicates the isostructural nature of their crystalline forms. The higher crystallinity of K4−2xMnxRe6Se8 colloids is followed by the bigger size of their nanocrystallites being within 20-42 nm ( Figure S4, Table S2 in ESI), which agrees well with the size distribution from TEM analysis (Figure 4). The size-values evaluated for K4−2xMnxRe6S8 and K4−2xMnxRe6Te8 from PXRD data are 11-16 and 7-12 nm, respectively (Figures S5 and S6,  Tables S2 and S3), which also close to the size-values revealed by the TEM analysis ( Figure  4). The PXRD pattern of K4−2xMnxRe6Te8 crystallites indicates that their diffraction pattern is closer to that of nanostructured systems (Figure 4d) due to the low crystallizing ability of K4−2xMnxRe6Te8 and very small crystallite sizes. However, a comparison of the positions of the observed interference peaks allows to state that the crystals of K4−2xMnxRe6Te8 colloids are not isostructural to the sulfide-and selenide-counterparts. It is worth noting that  , Tables S2 and S3), which also close to the size-values revealed by the TEM analysis ( Figure 4). The PXRD pattern of K 4−2x Mn x Re 6 Te 8 crystallites indicates that their diffraction pattern is closer to that of nanostructured systems (Figure 4d) due to the low crystallizing ability of K 4−2x Mn x Re 6 Te 8 and very small crystallite sizes. However, a comparison of the positions of the observed interference peaks allows to state that the crystals of K 4−2x Mn x Re 6 Te 8 colloids are not isostructural to the sulfide-and selenide-counterparts. It is worth noting that the peculiar crystal packing was also revealed for the gadolinium complexes of [{Re 6 Te 8 (CN) 6 ] 4− cluster [42].
PXRD analysis of dried colloids reveals different extent of crystallinity of the colloids (Figure 4). The similarity in the PXRD patterns of K4−2xMnxRe6S8 and K4−2xMnxRe6Se8 colloids indicates the isostructural nature of their crystalline forms. The higher crystallinity of K4−2xMnxRe6Se8 colloids is followed by the bigger size of their nanocrystallites being within 20-42 nm ( Figure S4, Table S2 in ESI), which agrees well with the size distribution from TEM analysis (Figure 4). The size-values evaluated for K4−2xMnxRe6S8 and K4−2xMnxRe6Te8 from PXRD data are 11-16 and 7-12 nm, respectively (Figures S5 and S6,  Tables S2 and S3), which also close to the size-values revealed by the TEM analysis ( Figure  4). The PXRD pattern of K4−2xMnxRe6Te8 crystallites indicates that their diffraction pattern is closer to that of nanostructured systems (Figure 4d) due to the low crystallizing ability of K4−2xMnxRe6Te8 and very small crystallite sizes. However, a comparison of the positions of the observed interference peaks allows to state that the crystals of K4−2xMnxRe6Te8 colloids are not isostructural to the sulfide-and selenide-counterparts. It is worth noting that the peculiar crystal packing was also revealed for the gadolinium complexes of [{Re6Te8(CN)6] 4− cluster [42]. It is worth noting that the as-separated colloids being further redispersed in "pure" water suffer from the instability manifested by the precipitation within one hour, since the residual amounts of F-127 cannot provide the colloidal stability of K 4−2x Mn x Re 6 Q 8 . Thus, the separated K 4−2x Mn x Re 6 Q 8 -based NPs should be further dispersed in aqueous solutions of the triblock copolymers (F-127, F-68 or P-123) for the high colloid stability at both ambient (298 K) and physiological (310-318 K) temperature. The aqueous colloids of K 4−2x Mn x Re 6 Q 8 generate sextet in the ESR spectra ( Figure S7) with the linewidths ∆H = 21.5 G, hyperfine coupling constant a Mn = 95 G and g = 2.002, which is peculiar for Mn 2+ ions, while the linewidth values argue for the interionic interactions derived from the package of K 4−2x Mn x Re 6 Q 8 complexes into the NPs. The ESR spectral features of the NPs indicate their d 5 electronic structure confirming its prospect for paramagnetic enhancement of the magnetic relaxation of water protons in their aqueous dispersions.

Magnetic Relaxivity of K 4−2x Mn x Re 6 Q 8
The longitudinal and transverse relaxation rates of water protons are enhanced in the aqueous colloids of K 4−2x Mn x Re 6 Q 8 , and the rates exhibit linear increase with the concentration growth of K 4−2x Mn x Re 6 Q 8 ( Figure 5 and Figure S3). This confirms low aggregation of the as-prepared NPs and allows to calculate the r 1 and r 2 values. It is worth noting that the linearity is observed for the rates measured at both 298 and 310 K, and the r 1 (2) values measured at 310 K are greater than those measured at 298 K. The observed temperature-induced relaxation enhancements refer to the condition T 1 < τ M , where the relaxivity enhanced by the factor τ R is retarded by the slow water exchange process [46]. This agrees well with the nanoparticulate form of K 4−2x Mn x Re 6 Q 8 complexes, which is the reason for a relaxivity enhancement due to long molecular reorientational time τ R . However, the supramolecular packing of K 4−2x Mn x Re 6 Q 8 complexes into the NPs can restrict the accessibility of Mn 2+ -centers to efficient exchange of the inner-sphere water molecules with the bulk. It is well-known that the water exchange is most efficient for the interfacial complexes, thus, surface-to-volume ratio should be of great impact on the relaxivity. Also, it is well-known that ultra-small size below 10 nm provides more efficient paramagnetic enhancement of water protons than that derived from the greater sized NPs [47]. However, the r 1 and r 2 values are the greatest for K 4−2x Mn x Re 6 Se 8 vs. those for K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Te 8 , which disagrees with the size values of the corresponding NPs evaluated from both PXRD and TEM measurements. In turn, the average size values measured for the NPs in the F-127 based solutions by the DLS technique indicate their aggregation (Table 1). Thus, the aggregation event can significantly level the deviations arisen from the different initial size of the NPs.  The r1 and r2 values of K4−2xMnxRe6Se8 dispersed in the solutions of different triblock copolymers depend on their nature, being the smallest for P-123, while growing in the following series P-123 < F-68 < F-127 (Table 1). The oxidation state of the interfacial Mn 2+ ions can change to Mn 3+ in the ambient conditions without deoxygenation, which is commonly observed for MnO-based NPs [47], but the oxidation state cannot be the reason for the tendency. It is worth assuming that the tendency derives from the different participation of the triblock copolymer molecules in hydrophilic coating of K4−2xMnxRe6Q8, although the average size values measured at 298 K reveal poor dependence on the triblock copolymer's nature. The lowest r1 and r2 values observed in the solutions of P-123 argues The r 1 and r 2 values of K 4−2x Mn x Re 6 Se 8 dispersed in the solutions of different triblock copolymers depend on their nature, being the smallest for P-123, while growing in the following series P-123 < F-68 < F-127 (Table 1). The oxidation state of the interfacial Mn 2+ ions can change to Mn 3+ in the ambient conditions without deoxygenation, which is commonly observed for MnO-based NPs [47], but the oxidation state cannot be the reason for the tendency. It is worth assuming that the tendency derives from the different participation of the triblock copolymer molecules in hydrophilic coating of K 4−2x Mn x Re 6 Q 8 , although the average size values measured at 298 K reveal poor dependence on the triblock copolymer's nature. The lowest r 1 and r 2 values observed in the solutions of P-123 argues for the lowest accessibility of the Mn 2+ ions to hydration, while the highest r 1 and r 2 values in the F-127based solution correlate with the most efficient hydrophilic coating of K 4−2x Mn x Re 6 Q 8 by F-127. It is also worth noting that the deviation between the r 1 and r 2 values arisen from the size and composition of the cores is on the same level of magnitude with the deviations between the values measured for different triblock copolymers. The aforesaid allows to hypothesize at least two types of the aggregation modes of K 4−2x Mn x Re 6 Q 8 in the solutions of F-127 and P-123 as it is schematically illustrated in the cartoon image ( Figure 6). The first type represented by the agglomerated NPs coated by the total hydrophilic shell (Figure 6a) in the greater extent restricts an accessibility of the Mn 2+ ions to hydration than the second one manifested by a self-assembly of the hydrophilic NPs (Figure 6b). Actually, both modes of the aggregation can be characterized by the similar sizes, but diverse r 1 and r 2 values ( Table 1). The predominance of the second aggregation mode correlates with the ability of the triblock copolymer to form a hydrophilic coating of the NPs, being the most for F-127. The r2/r1 ratios are greatly affected by the hydration number (q) of Mn(II) complexes, since longitudinal and transverse relaxation rates are differently contributed by the scalar mechanism [14]. Thus, r2/r1 ratio is the greatest (4.8) for Mn(II) aqua ions, while comes to minimum (1.2) when q = 0 [48]. The ratios for K4−2xMnxRe6S8 and K4−2xMnxRe6Se8 lie within 1.21-1.24 in the solutions of the triblock copolymers independently on their nature, while the greater ratios are revealed for K4−2xMnxRe6Te8 ( Table 1). The tendency agrees well with the PXRD data ( Figure 4) revealing isostructural features of K4−2xMnxRe6S8 and K4−2xMnxRe6Se8, while the ratios 1.66-1.73 of K4−2xMnxRe6Te8 correlate with its structural specificity. Thus, the structural features of K4−2xMnxRe6Q8 significantly affect the r2/r1 ratio, while their effect on the r1 and r2 values is distorted by the influence of the aggregation behavior of K4−2xMnxRe6Q8 in the aqueous solutions.
The r1 values of F-127-K4−2xMnxRe6Se8 colloids are greater than those reported for the Gd-containing commercial CAs, while the r2/r1 values of the colloids are below 2 (Table 1) similar with the Gd-containing CAs [47]. Since the magnetic field strengths also influence both r1(2) and r2/r1 values of nanoparticulate CAs [47], the literature values measured at 0.5 T are required for the correct comparison with the r2/r1 values collected in Table 1. The comparison indicates that the represented r2/r1 values are the least among those reported for the Mn-containing nanoparticulate CAs at 0.5 T [49,50]. The measurements at 1.5 T by means of the whole-body scanner reveal the following relaxivity values r1(2) = 6.5(12.8) mM −1 s −1 and r2/r1 = 1.96 for F-127-K4−2xMnxRe6Se8. The values measured for Omniskan by the same equipment (r1(2) = 3.7(4.5) mM −1 s −1 ) agree well with the literature values [51] (Figure 7). The comparison of the latter values with those of F-127-K4−2xMnxRe6Se8 colloids reveals their advantage vs. the commercial Gd-containing CAs. The r1(2) values of F-127- The r 2 /r 1 ratios are greatly affected by the hydration number (q) of Mn(II) complexes, since longitudinal and transverse relaxation rates are differently contributed by the scalar mechanism [14]. Thus, r 2 /r 1 ratio is the greatest (4.8) for Mn(II) aqua ions, while comes to minimum (1.2) when q = 0 [48]. The ratios for K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 lie within 1.21-1.24 in the solutions of the triblock copolymers independently on their nature, while the greater ratios are revealed for K 4−2x Mn x Re 6 Te 8 ( Table 1). The tendency agrees well with the PXRD data ( Figure 4) revealing isostructural features of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 , while the ratios 1.66-1.73 of K 4−2x Mn x Re 6 Te 8 correlate with its structural specificity. Thus, the structural features of K 4−2x Mn x Re 6 Q 8 significantly affect the r 2 /r 1 ratio, while their effect on the r 1 and r 2 values is distorted by the influence of the aggregation behavior of K 4−2x Mn x Re 6 Q 8 in the aqueous solutions.
The r 1 values of F-127-K 4−2x Mn x Re 6 Se 8 colloids are greater than those reported for the Gd-containing commercial CAs, while the r 2 /r 1 values of the colloids are below 2 (Table 1) similar with the Gd-containing CAs [47]. Since the magnetic field strengths also influence both r 1(2) and r 2 /r 1 values of nanoparticulate CAs [47], the literature values measured at 0.5 T are required for the correct comparison with the r 2 /r 1 values collected in Table 1. The comparison indicates that the represented r 2 /r 1 values are the least among those reported for the Mn-containing nanoparticulate CAs at 0.5 T [49,50]. The measurements at 1.5 T by means of the whole-body scanner reveal the following relaxivity values r 1(2) = 6.5(12.8) mM −1 s −1 and r 2 /r 1 = 1.96 for F-127-K 4−2x Mn x Re 6 Se 8 . The values measured for Omniskan by the same equipment (r 1(2) = 3.7(4.5) mM −1 s −1 ) agree well with the literature values [51] (Figure 7). The comparison of the latter values with those of F-127-K 4−2x Mn x Re 6 Se 8 colloids reveals their advantage vs. the commercial Gd-containing CAs. The r 1 (2) values of F-127-K 4−2x Mn x Re 6 Se 8 are close to the best reported in literature values of Mn-containing CAs (r 1(2) = 8.4(16.8) mM −1 s −1 and r 2 /r 1 = 2.0) measured at 1.5 T [47], however, the r 2 /r 1 values of the Mn-containing nanoparticulate CAs measured at magnetic field strength greater than 1.5 T are far above 2.0 [10,52,53]. The level of r 2 /r 1 about 2 is convenient for the low interference of T 1 -weighted relaxivity by the T 2 -weighted one [47].

Leaching, Cytotoxicity, Hemagglutination Assay and Imaging Capacity of K4−2xMnxRe6Se8
The leaching of Mn 2+ ions from K4−2xMnxRe6Se8 buffered solutions of BSA modeling blood plasma can be easily monitored through r2/r1 ratio, since the ratio for aqua Mn 2+ ions is ~4.8 [48], which is substantially higher than that of the NPs in aqueous solutions. Thus, the remaining unchanged of the r2/r1 ratio of K4−2xMnxRe6Se8 in the buffered solutions within ten days (Figure 6c) indicates the insignificant (below 5% of the total concentration of Mn) leaching of Mn 2+ ions from the NPs. The invariance of the DLS measurements of F-127-K4−2xMnxRe6Se8 colloids performed within one month ( Figure S8) confirms their chemical and colloidal stability.
The insignificant leaching well correlates with no effect of K4−2xMnxRe6S8 and K4−2xMnxRe6Se8 on the cell viability of both M-HeLa and Chang Liver cell lines (Table S5), where the cell viability values does not reach 50% even at 90 μM of the NPs.
Intravenous applicability of CAs raises a question about their hemocompatibility [54]. The hemagglutination assay has been performed to reveal hemocompatibility of K4−2xMnxRe6S8 and K4−2xMnxRe6Se8 at the concentrations varying from 10 to 100 μM. It can be seen from Figure 8a that a round red button is present at the bottom of the wells filled by erythrocytes in physiological saline and mixed with the colloids, which corresponds to a negative reaction, while a carpetlike structure is peculiar for agglutinating erythrocytes (positive control). This indicates the lack of hemagglutination even at the greatest concen- The leaching of Mn 2+ ions from K 4−2x Mn x Re 6 Se 8 buffered solutions of BSA modeling blood plasma can be easily monitored through r 2 /r 1 ratio, since the ratio for aqua Mn 2+ ions is~4.8 [48], which is substantially higher than that of the NPs in aqueous solutions. Thus, the remaining unchanged of the r 2 /r 1 ratio of K 4−2x Mn x Re 6 Se 8 in the buffered solutions within ten days (Figure 6c) indicates the insignificant (below 5% of the total concentration of Mn) leaching of Mn 2+ ions from the NPs. The invariance of the DLS measurements of F-127-K 4−2x Mn x Re 6 Se 8 colloids performed within one month ( Figure S8) confirms their chemical and colloidal stability.
The insignificant leaching well correlates with no effect of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 on the cell viability of both M-HeLa and Chang Liver cell lines (Table S5), where the cell viability values does not reach 50% even at 90 µM of the NPs.
Intravenous applicability of CAs raises a question about their hemocompatibility [54]. The hemagglutination assay has been performed to reveal hemocompatibility of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 at the concentrations varying from 10 to 100 µM. It can be seen from Figure 8a that a round red button is present at the bottom of the wells filled by erythrocytes in physiological saline and mixed with the colloids, which corresponds to a negative reaction, while a carpetlike structure is peculiar for agglutinating erythrocytes (positive control). This indicates the lack of hemagglutination even at the greatest concentrations of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 .

Conclusions
Summarizing, the complex formation of Mn 2+ ions with [{Re6Q8}(CN)6] 4− (Re6Q8, Q = S 2− , Se 2− or Te 2− ) is for the first time represented as the facile route to generate heterometallic nanoparticles K4−2xMnxRe6Q8, where x = 1.3-1.8. The results highlighted the temperature-dependent aggregation behavior of the triblock copolymers (F-127, F-68 and P-123) as the key factor for stabilizing of K4−2xMnxRe6Q8 colloids in ambient conditions and their phase separation under the centrifugation. The F-127 fits well to the aforesaid requirements, thus, the treating by F-127 of K4−2xMnxRe6Q8 colloids allows to gain in high colloid stability and facile phase separation. The variation of the ligand's structure revealed the [{Re6Se8}(CN)6] 4− cluster complex as the optimal one for a combination of the magnetic relaxivity values (r1 = 8.9 and r2 = 10.9 mM −1 s −1 at 0.47 T and 310 K) with the low levels of hemoagglutination activity and cytotoxicity of the colloids. Thus, the reported herein r1 and r2 values of F-127-K4−2xMnxRe6Q8 colloids are not among the leaders, but their r2/r1 values are within 1.2-1.7, which is the advantage of the colloids differentiating them from the documented in literature nanoparticulate Mn-containing CAS. Thus, both molecular and nano-structural optimization of K4−2xMnxRe6Q8 colloids was successful for optimal balance of high T1-and T2-weighted contrast ability with an applicability for in vivo imaging of the colloids derived from their low hemoagglutination and cytotoxicity.  Table S1: XO-assisted and ICP-OES analysis of K4-2xMnxRe6Q8-based hydrophilic NPs; Figure S3: IR spectra of K4-2xMnxRe6Q8based NPs and [Re6Q8(CN)6] 4− ; Figure S4: Powder XRD pattern of K4-2xMnxRe6Q8, Q = Te 2− complex, experimental (blue), calculated (red) and their difference (grey) curves are depicted; Figure S5: Powder XRD pattern of K4-2xMnxRe6Q8, Q = Se 2− complex, experimental (green), calculated (red) and their The microscopic images of the erythrocytes mixed with the aqueous colloids of K 4−2x Mn x Re 6 S 8 and K 4−2x Mn x Re 6 Se 8 at the highest colloid concentration demonstrated in Figure 8b also reveal very poor agglutination if any. It is worth explaining the low hemagglutination activity of the F-127-K 4−2x Mn x Re 6 Q 8 colloids by the hydrophilic PEO chains constituting their exterior coating. This, in turn, is a good prerequisite for a long blood circulation half-life time under in vivo application of the colloids.

Conclusions
Summarizing, the complex formation of Mn 2+ ions with [{Re 6 Q 8 }(CN) 6 ] 4− (Re 6 Q 8 , Q = S 2− , Se 2− or Te 2− ) is for the first time represented as the facile route to generate heterometallic nanoparticles K 4−2x Mn x Re 6 Q 8 , where x = 1.3-1.8. The results highlighted the temperature-dependent aggregation behavior of the triblock copolymers (F-127, F-68 and P-123) as the key factor for stabilizing of K 4−2x Mn x Re 6 Q 8 colloids in ambient conditions and their phase separation under the centrifugation. The F-127 fits well to the aforesaid requirements, thus, the treating by F-127 of K 4−2x Mn x Re 6 Q 8 colloids allows to gain in high colloid stability and facile phase separation. The variation of the ligand's structure revealed the [{Re 6 Se 8 }(CN) 6 ] 4− cluster complex as the optimal one for a combination of the magnetic relaxivity values (r 1 = 8.9 and r 2 = 10.9 mM −1 s −1 at 0.47 T and 310 K) with the low levels of hemoagglutination activity and cytotoxicity of the colloids. Thus, the reported herein r 1 and r 2 values of F-127-K 4−2x Mn x Re 6 Q 8 colloids are not among the leaders, but their r 2 /r 1 values are within 1.2-1.7, which is the advantage of the colloids differentiating them from the documented in literature nanoparticulate Mn-containing CA S . Thus, both molecular and nano-structural optimization of K 4−2x Mn x Re 6 Q 8 colloids was successful for optimal balance of high T 1 -and T 2 -weighted contrast ability with an applicability for in vivo imaging of the colloids derived from their low hemoagglutination and cytotoxicity.  Table S1: XO-assisted and ICP-OES analysis of K 4−2x Mn x Re 6 Q 8 -based hydrophilic NPs; Figure S3: IR spectra of K 4−2x Mn x Re 6 Q 8 -based NPs and [Re 6 Q 8 (CN) 6 ] 4− ; Figure S4: Powder XRD pattern of K 4−2x Mn x Re 6 Q 8 , Q = Te 2− complex, experimental (blue), calculated (red) and their difference (grey) curves are depicted; Figure S5: Powder XRD pattern of K 4−2x Mn x Re 6 Q 8 , Q = Se 2− complex, experimental (green), calculated (red) and their difference (grey) curves are depicted; Figure S6: Powder XRD pattern of K 4−2x Mn x Re 6 Q 8 , Q = S 2− complex, experimental (black), calculated (red) and their difference (grey) curves are depicted; Table S2: Crystallite size, calculated from experimental diffraction patterns of K 4−2x Mn x Re 6 Q 8 , Q = Se 2− ; Table S3: Crystallite size, calculated from experimental diffraction patterns of K 4−2x Mn x Re 6 Q 8 , Q = S 2− complex; Table S4: Crystallite size, calculated from experimental diffraction patterns of K 4−2x Mn x Re 6 Q 8 , Q = Te 2− ; Figure S7: ESR spectra of of F-127-K 4−2x Mn x Re 6 Q 8 : Q = S 2− (a); Q = Se 2− (b); Q = Te 2− (c); Figure S8: Size distribution by volume of F-127-K 4−2x Mn x Re 6 Q 8 at initial time (black line) and after 7 day (red line); Table S5: Cell viability of F-127-K 4−2x Mn x Re 6 Q at different manganese concentrations. References [55,56] are cited in the supplementary materials.